The human immune system responds to viral infections by generating antibodies to various viral antigens. The number of antibodies and the amount of each different antibody produced depends on the virus and the virus antigen which initiates the antibody response.
Current immunoassay systems have proven useful to monitor viral infections. For example, it is possible to monitor HIV-1 or HIV-2 infections using immunoassays which use partially purified viral lysates. Unfortunately, these lysates may contain disproportionate quantities of various antigens or may lack important antigenic polypeptides. These deficiencies detrimentally affect both the sensitivity and selectivity of the assay. For HIV viruses, vital lysates may lack the transmembrane peptide, gp41, and the outer envelope protein, gp120.
Viral lysates are also unsatisfactory because they contain undesired immunologically reactive cellular components, antigenic aggregates and degraded antigenic fragments, as well as having antigenic peptides with differing degrees of glycosylation. These unavoidable extrinsic factors create purification problems when the viral lysates are separated on a gel because the resolution is less than ideal. Less than ideal resolution magnifies the separation difficulties caused by missing bands, diffused bands, overlapping nonspecific bands or too many bands and leads to unsatisfactory immunoblots that are difficult to interpret when the gel is transferred to an immunoblot surface. Furthermore, it is often difficult to prepare sufficient quantities of partially purified viral lysates for traditional immunoblots and the lack of sufficient quantities of a desired antigen increases the purification problems.
The problems associated with using viral lysates can be partially avoided by expressing desired antigenic polypeptides in various heterologous cell systems. These systems use recombinant techniques to insert virion genes of interest in cells that can produce quantities of these polypeptide products. This procedure often results in the production of the desired antigen in sufficient quantities, but is typically associated with the presence of new impurities and degraded antigenic fragments. Antigens produced by these methods may also form aggregates that must be minimized by using reducing agents or require the use of multiple gels to avoid both aggregation and degradation problems.
Traditional immunoblot methods lack the versatility to analyze multiple samples for reactivity with multiple antigens because of the need for many purification steps to provide antigens of a sufficient degree of homogeneity needed for selective and sensitive assays. The typical Western blot method is described by Gordon et al., U.S. Pat. No. 4,452,901 filed Mar. 18, 1980 issued Jun. 5, 1984. The document describes the procedures necessary to transfer antigenic peptides from a single gel to nitrocellulose. The present invention teaches the transfer of antigenic polypeptides from multiple gels.
The use of several antigens in a single assay has been described. Lin et al., J. Virol. 59:522-24 (1986) describes a dual antibody probing technique that permitted identification of Epstein-Barr virus and different herpes virus antigens in the same Western blot.
It is also possible to use multiple detection methods on a single Western blot. Lee et al., J. Immuno. Methods, 106:27-30 (1988) describes a technique which uses sequentially applied sets of probing antibodies, enzyme-conjugated developing antibodies and enzyme substrates to detect two or more types of interferon on a single Western blot. The same result can be achieved by simultaneously applying more than one type of probing antibody using a mixture of different enzyme-conjugated developing antibodies followed by successive applications of different substrates.
Gordon et al., European Patent Application Publication Number 0 063 810 published Mar. 11, 1982 describes immunoassay devices and kits made up of antigens or antibodies or both bound to a solid support. The use of the described solid supports makes possible a number of simultaneous antibody-antigen reactions in one operation. Gordon et al. describes applying single or successive doses of solutions of antigens or antibodies to the surface of the solid support using a pipet or syringe. In a preferred embodiment, the antigen is applied as a microdot formed by adding small volumes of an antigenic solution. The application does not describe immunoassays using antigens purified from a complex mixture of proteins.
Lefkovits, WO 87/03965, published Jul. 2, 1987, describes a test strip for several simultaneous assays. The test strip is made from nitrocellulose impregnated with an antibody that is cut into strips and mounted on an inert backing. Lefkovits describes soaking a support sheet in a solution of antigenic peptide and then cutting the sheet into strips but does not describe transferring the antigen to the solid support electrophoretically.